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protein indentification


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#1 madmaxx

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Posted 26 April 2010 - 03:54 AM

hello everyone, i am trying to purify a protein of 230 kda using preparative sds page 3.5%-12% ,using muscle homogenate as source. the region i cut on the preparative gel ,for my protein,is according to rf of mol weight. when i try to identify the protein using antibodies on western blot they migrate to lower region with less rf values than the one i had cut. pls help. :) . the protein that i have isolated is 185.5 and not 230 kda. why is so.....

does the migration of protein in group differ from when it is alone on the gel????

#2 Prep!

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Posted 28 April 2010 - 01:04 AM

i presume u are using the same marker system in both the preparative gel and the western blot... i m not suer as i have never done a preparative scale SDS-PAGE but may be over loading with very high amounts retards the migration??!! (tat is the band is not compact and so the Rf calculation is error prone!!?!)

Edited by Prep!, 28 April 2010 - 01:08 AM.

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#3 Inmost sun

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Posted 29 April 2010 - 11:06 AM

hello everyone, i am trying to purify a protein of 230 kda using preparative sds page 3.5%-12% ,using muscle homogenate as source. the region i cut on the preparative gel ,for my protein,is according to rf of mol weight. when i try to identify the protein using antibodies on western blot they migrate to lower region with less rf values than the one i had cut. pls help. :) . the protein that i have isolated is 185.5 and not 230 kda. why is so.....

does the migration of protein in group differ from when it is alone on the gel????


differences in predicted molecular mass may result from proteolysis, or posttranslational modifications. or as suggested, unprecise reference proteins




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